Epithelial cells, which line both the inside cavities and outside of the body, exist in tissues as monolayers, multilayers of cells, and three dimensional tube/duct structures. Proper formation and homeostasis of the epithelium is critical for tissue and organ function; dysregulation of the epithelium is associated with epithelial barrier loss (including sepsis), defective wound healing, and development and progression of cancer. Although mechanical forces on epithelial cells have been shown to influence cell organization, proliferation, and migration, it is not known the mechanisms by which cells respond to force. This proposal examines the role of force across proteins in both cell-cell junctions and the nuclear linker of nucleoskeleton to cytoskeleton (LINC) complex as mediators of epithelial homeostasis. Strong cell-cell junctions are critical to the integrity of the epithelium, including cell cohesion, barrier function, and ability to resist mechanical stress. Loss of junctions is associated with epithelial dysfunction including inflammatory-induced increases in permeability and epithelial to mesenchymal transition (EMT). Although formation cell-cell adhesions have been shown to be critical regulators of cell proliferation, migration, and tissue organization, very little is known how cell-cell junction forces contribute to these processes. In addition to altering junction forces, externally applied forces are likely transmitted inside the cell, across the cytoskeleton, and onto organelles. The nucleus, which is physically connected to the cytoskeleton by the LINC complex, is likely affected by external forces. Nuclear forces have been suggested to regulate nuclear geometry and nuclear positioning, both of which are altered in a variety of diseases, including cancer. The major research goals of this MIRA proposal are to examine how forces across cell-cell junction proteins and the nuclear LINC complex regulate epithelial proliferation, migration, junction stability, and 3D organization. Novel FRET-based tension biosensors will be used to directly measure forces across tight junctions, adherens junctions, and desmosomes at cell-cell junctions and specific isoforms of nesprin at the LINC complex. Treatments or mutants for which junction or nuclear force is perturbed will be used to assess the causal nature of force in regulation of the epithelium. An additional goal of this proposal is to identify the relationship between forces across cell-cell junctions, cell-matrix adhesions, and the nuclear LINC complex, identifying how forces are transmitted from one region of the cell to another. This comprehensive study of cell-cell, cell-matrix, and nuclear forces will greatly advance the understanding of epithelial homeostasis, which includes the processes of wound repair, inflammation, and epithelial tissue development and organization, as well as epithelial diseases, including cancer, fibrosis, and chronic inflammation. Junction and nuclear forces may represent a universal mechanism to regulate proliferation, migration, and organization; therefore, results from this study may also be relevant to a large number of non- epithelial cells and tissues.